Construction of a derivative of Tn917 containing an outward-directed promoter and its use in Bacillus subtilis

Zagorec, Monique; Steinmetz, Michel O.

doi:10.1099/00221287-137-1-107

Cited by 22 publications

(12 citation statements)

References 23 publications

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“…Transposon insertions most commonly result in null loss-of-function mutations in the gene into which they have inserted. We set out to build a transposon which, when integrated, would cause the high-level expression of the adjacent gene(s) for use in artificial-expression "gene-trap" screens (33). To build transposons with an outwardoriented, strong, constitutive promoter, the P hyspank promoter was PCR amplified using primers 1886/2271 and pDR111 as the DNA template (3).…”

Section: Resultsmentioning

confidence: 99%

“…As proof of principle, we generated transposons with kanamycin, spectinomycin, and chloramphenicol resistance cassettes. We further generated a transposon called TnHyJump with an outward-facing, unregulated, high-expression promoter (P hyspank ) and a transposon called TnLacJump, with a promoterless lacZ gene for generating random ␤-galactosidase transcriptional reporters (24,33). The system presented here expands the kind of genetic screens that can be conducted with mariner in terms of antibiotic resistance cassettes, reporters, and many other constructs for use not only in B. subtilis but all mariner-compatible bacteria.…”

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confidence: 99%

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Modified mariner Transposons for Random Inducible-Expression Insertions and Transcriptional Reporter Fusion Insertions in Bacillus subtilis

Pozsgai

Blair

Kearns

2012

Appl Environ Microbiol

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Transposons are mobile genetic elements bounded by insertion sequences that are recognized by a specific mobilizing transposase enzyme. The transposase may mobilize not only the insertion sequences but also intervening DNA. mariner is a particularly efficient transposon for the random chromosomal integration of genes and insertional mutagenesis. Here, we modify an existing mariner transposon, TnYLB, such that it can easily be genetically manipulated and introduced into Bacillus subtilis. We generate a series of three new mariner derivatives that mobilize spectinomycin, chloramphenicol, and kanamycin antibiotic resistance cassettes. Furthermore, we generate a series of transposons with a strong, outward-oriented, optionally isopropyl-␤-Dthiogalactopyranoside (IPTG)-inducible promoter for the random overexpression of neighboring genes and a series of transposons with a promoterless lacZ gene for the random generation of transcriptional reporter fusions. We note that the modification of the base transposon is not restricted to B. subtilis and should be applicable to any mariner-compatible host organism, provided that in vitro mutagenesis or an in vivo species-specific delivery vector is employed.

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Section: Resultsmentioning

confidence: 99%

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confidence: 99%

Modified mariner Transposons for Random Inducible-Expression Insertions and Transcriptional Reporter Fusion Insertions in Bacillus subtilis

Pozsgai

Blair

Kearns

2012

Appl Environ Microbiol

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“…GM982, a sacY-overexpressing GM904 derivative, was obtained by replacing the 1-kilobase-pair Sal I-Sst I segment containing the 5' end of sacX (3) in the GM904 chromosome with the aphA' cassette, a kanamycin-resistance gene devoid of transcription terminator (11); this substitution created an Abbreviation: RAT, ribonucleic antiterminator. *To whom reprint requests should be addressed.…”

Section: Methodsmentioning

confidence: 99%

Specificity determinants and structural features in the RNA target of the bacterial antiterminator proteins of the BglG/SacY family.

Aymerich

Steinmetz²

1992

Proc. Natl. Acad. Sci. U.S.A.

123

157

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Induction of the Bacillus subtilis sacB gene and sacPA operon and Escherichia coli bgl operon is mediated by structurally homologous antiterminators encoded by the sacY, sacT, and bhgG genes, respectively. When activated, these proteins prevent early transcription termination at terminators located in the leader regions of the three operons. BglG was previously shown to bind in vitro to an imperfectly palindromic 29-nucleotide RNA sequence located upstream of the terminator and partially overlapping with it [Houman, F., DiazTorres, M. R. & Wright, A. (1990) Cell 62, 1153-11631. Similar motifs, here termed ribonucleic antiterminators (RATs), strongly conserved in sequence and in position, are found in the leader of both sacB and sacPA. Mutations were created in sacB RAT and tested in B. subtilis; this showed that sacB RAT is the target for SacY-mediated induction of sacB and that a stem-oop structure in the mRNA is required for regulatory function. Mutations increasing the similarity of the sacB RAT with those of sacPA or bgl rendered sacB inducible by SacT or BglG, respectively; most of these changes did not strongly affect induction by SacY, suggesting that the nucleotides at these variable positions act as negative specificity determinants.Induction of the Bacillus subtilis levansucrase (sacB) gene by sucrose and the Escherichia coli f3-glucoside (bgt) operon by (3-glucosides is controlled by similar transcriptional antitermination mechanisms (1-3). In both systems, transcription initiates constitutively from the promoter; in the absence of inducer, most transcripts terminate at a terminator located in the leader region, between the promoter and the first gene; in the presence of inducer, a trans-acting protein (antiterminator) is activated and prevents early transcription termination (1, 2). Sucrose induction of the B. subtilis sucrase (sacPA) operon appears to follow a similar mechanism (4, 5). The antiterminator BglG is encoded by the first gene of the bgl operon. Between bglG and the second gene (bglF) lies a second BglG-controlled terminator (6). In B. subtilis, the sac Y and sacT gene products are required for induction of sacB and sacPA, respectively (3-5). BglG, SacY, and SacT are very similar proteins, with 48% identity between SacY and SacT and 30%1o between SacY and BglG (5). It was shown in vitro that BglG is an RNA-binding protein that recognizes a specific sequence located upstream of each of the two bgl conditional terminators (7). These 29-nucleotide sequences partially overlap with the terminators; thus, binding of BglG to these sequences, hereafter referred to as ribonucleic antiterminator (RAT) sequences, might prevent the formation ofthe termination structures. Similar RAT sequences are present in the sacB and sacPA leader regions and overlap with the terminator sequences in exactly the same way (5). This suggests that they are binding sites for SacY and SacT in the leader of sacB and sacPA mRNAs, respectively. The same model can therefore be proposed for the three systems: when ac...

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“…The presence of genes homologous to those encoding maltodextrin transporters suggested that oligosaccharide products resulting from the activity of an extracellular enzyme were being imported as substrates for the intracellular ␤-galactosidases. An example of this gene arrangement is found in Bacillus subtilis surrounding lacA (BSU34130) (12,14), one of two nonadjacent GHF 42-encoding genes (the other being yesZ [BSU07080] [70]). We therefore used B. subtilis as a model to explore our hypothesized function.…”

Section: -Linked Galactosidic Substrate O-nitrophenyl-␤-d-galactopyramentioning

confidence: 99%

Bioinformatic, Genetic, and Biochemical Evidence that Some Glycoside Hydrolase Family 42 β-Galactosidases Are Arabinogalactan Type I Oligomer Hydrolases

Shipkowski

Brenchley

2006

Appl Environ Microbiol

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Glycoside hydrolases are organized into glycoside hydrolase families (GHFs) and within this larger group, the ␤-galactosidases are members of four families: 1, 2, 35, and 42. Most genes encoding GHF 42 enzymes are from prokaryotes unlikely to encounter lactose, suggesting a different substrate for these enzymes. In search of this substrate, we analyzed genes neighboring GHF 42 genes in databases and detected an arrangement implying that these enzymes might hydrolyze oligosaccharides released by GHF 53 enzymes from arabinogalactan type I, a pectic plant polysaccharide. Because Bacillus subtilis has adjacent GHF 42 and GHF 53 genes, we used it to test the hypothesis that a GHF 42 enzyme (LacA) could act on the oligosaccharides released by a GHF 53 enzyme (GalA) from galactan. We cloned these genes, plus a second GHF 42 gene from B. subtilis, yesZ, into Escherichia coli and demonstrated that cells expressing LacA with GalA gained the ability to use galactan as a carbon source. We constructed B. subtilis mutants and showed that the increased ␤-galactosidase activity generated in response to the addition of galactan was eliminated by inactivating lacA or galA but unaffected by the inactivation of yesZ. As further demonstration, we overexpressed the LacA and GalA proteins in E. coli and demonstrated that these enzymes degrade galactan in vitro as assayed by thin-layer chromatography. Our work provides the first in vivo evidence for a function of some GHF 42 ␤-galactosidases. Similar functions for other ␤-galactosidases in both GHFs 2 and 42 are suggested by genomic data.␤-Galactosidases, such as the LacZ ␤-galactosidase of Escherichia coli, are used as molecular biology tools, industrial enzymes, and models for exploring structure-function relationships. However, these uses do not often yield insight into the physiological roles of these enzymes. Although ␤-galactosidases (EC 3.2.1.23) are also found in glycoside hydrolase families (GHFs) 1, 2, and 35 (groups differing in secondary structure and other attributes [20,21]), we were especially curious about the in vivo function of GHF 42 ␤-galactosidases because they occur in organisms isolated from diverse habitats where lactose would not be present, e.g., hot springs (33, 47, 65), hypersaline environments (27, 54), and soil (Bacillus and Streptomyces spp.).Over a dozen GHF 42 enzymes have been characterized, and most are ␤-galactosidases (as shown by maximal activity with the) with less than 10% relative activity on nongalactosidic chromogens (with two exceptions [27,34]). However, experimental data supporting lactose hydrolysis by GHF 42 ␤-galactosidases are absent (34, 44, 48) or weak. Several prokaryotes possessing a GHF 42 gene are unable to grow on lactose as a sole carbon source (1, 9, 27), and at least two GHF 42 ␤-galactosidases do not cleave lactose in vitro (27,64). The determination of growth on lactose can be complicated by the presence of multiple ␤-galactosidases (7,16,25) because not all of the ␤-galactosidases may be participating equally (or at all) as...

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Construction of a derivative of Tn917 containing an outward-directed promoter and its use in Bacillus subtilis

Cited by 22 publications

References 23 publications

Modified mariner Transposons for Random Inducible-Expression Insertions and Transcriptional Reporter Fusion Insertions in Bacillus subtilis

Modified mariner Transposons for Random Inducible-Expression Insertions and Transcriptional Reporter Fusion Insertions in Bacillus subtilis

Specificity determinants and structural features in the RNA target of the bacterial antiterminator proteins of the BglG/SacY family.

Bioinformatic, Genetic, and Biochemical Evidence that Some Glycoside Hydrolase Family 42 β-Galactosidases Are Arabinogalactan Type I Oligomer Hydrolases

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